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  Vol  5,  No  1  (2017)   ISSN  2167-­‐8677  (online)   DOI  10.5195/d3000.2017.78    

Fluctuating  dental  asymmetry  in  rabbits  with  familial  nonsyndromic   coronal  suture  synostosis   1

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Mae  A.  Hyre ,  Seth  M.  Weinberg ,  Gregory  M.  Cooper ,  James  Gilbert ,  Michael  I.  Siegel ,  Mark  P.  Mooney  

 

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Oro-­‐Maxillo-­‐Facial  Surgeon,  Private  Prac+ce,  Charleston  WV   University  of  Pi.sburgh,  School  of  Dental  Medicine   3 University  of  Pi.sburgh,  Department  of  Plas0c  Surgery 2

Abstract   Fluctua'ng   dental   asymmetry   has   been   linked   to   condi'ons   of   unstable   pre-­‐   and   peri-­‐natal   development.   Familial,   nonsyndromic  craniosynostosis  disrupts  early  craniofacial  development  through  localized  excessive  calvarial  ossifica-­‐ !on  leading  to  the  premature  fusion  of  the  calvarial  sutures.  Such  abnormal  gene  expression  may  also  produce  sys-­‐ temic   stress   resul+ng   in   developmental   instability,   thereby   affec+ng   normal   trait   symmetry.   The   present   study   was   designed  to  test  this  hypothesis  by  examining  fluctua4ng  dental  asymmetry  in  an  inbred  strain  of  rabbits  with  familial,   nonsyndromic  coronal  craniosynostosis.    The  mesiodistal  (MD)  and  buccolingual  (BL)  dimensions  of  the  right  and  le:   maxillary   first   molars   were   measured   in   four   groups   of   New   Zealand   white   rabbits   (N=176;   n=40   with   early-­‐onset   synostosis,   n=65   with   delayed-­‐onset   synostosis,   n=46   in-­‐colony,   phenotypically   normal   rabbits,   and   n=25   wild-­‐type   normal  controls).  For  each  variable,  raw  signed  asymmetry  was  calculated  (le:-­‐right)  and  tested  for  assump2ons  of   fluctua&ng   asymmetry   (i.e.,   normality   and   non-­‐direc&onality).   Any   group   that   did   not   meet   these   assump.ons   was   excluded  from  further  analysis.  Using  a  standard  size-­‐adjusted,  fluctua,ng  asymmetry  index,  mean  fluctua,ng  asym-­‐ metry  was  calculated  and  compared  across  groups  with  non-­‐parametric  sta+s+cs.    For  the  MD  dimension,  no  signifi-­‐ cant  (p  >  0.05)  group  differences  in  mean  fluctua8ng  asymmetry  were  observed  among  groups.  In  contrast,  rabbits   with  early-­‐onset  synostosis  had  significantly  (p  <  0.05)  more  fluctua;ng  asymmetry  in  the  BL  dimension  compared  to   wild-­‐type   controls.     Results   demonstrate   increased   fluctua1ng   dental   asymmetry   in   rabbits   with   nonsyndromic,   early-­‐ onset  coronal  suture  synostosis  and  suggest  that  the  molecular  events  producing  suture  synostosis  locally  may  have   also  have  systemic  effects.  Knowledge  of  these  systemic  interac/ons  may  contribute  to  a  fuller  understanding  of  the   phenotypic  spectrum  observed  in  individuals  with  nonsyndromic  craniosynostosis.  

Cita%on:  Hyre  M,  et  al.  (2017)  Fluctua'ng  dental   asymmetry  in  rabbits  with  familial  nonsyndromic   coronal  suture  synostosis.  Den$stry  3000.   1:a001  doi:10.5195/d3000.2017.78   Received:    July  11,  2017   Accepted:    August  18,  2017   Published:    October  10,  2017   Copyright:  ©2017  Hyre  M,  et  al.  This  is  an  open   access   ar!cle   licensed   under   a   Crea!ve   Com-­‐ mons   A"ribu"on   Work   4.0   United   States   Li-­‐ cense.   Email:  mpm4@pi&.edu

 

Introduction   Nonsyndromic,  simple  cranio-­‐ synostosis  is  a  relatively  rare  condi-­‐ tion  (0.06  –  1.9/1000)  that  occurs   when  one  or  more  of  the  calvarial   sutures  fuses  prematurely  during  the   period  of  active  brain  growth,  result-­‐ ing  in  aberrant  patterns  of  growth  in   both  the  neurocranium  and  viscer-­‐ ocranium  [1].  In  recent  years,  cranio-­‐ facial  biologists  have  begun  to  unrav-­‐ el  the  biological  pathways  underlying   abnormal  suture  morphogenesis.  The   emerging  picture  is  one  of  complexi-­‐ ty,  involving  multiple  osteogenic   growth  factors  and  genes  (FGFs,   TGFβs,  BMPs,  MSXs,  etc.)  operating  at   various  levels  of  regulation  [2,3].   While  the  genetic  mutations  respon-­‐ sible  for  syndromic  cases  of  cranio-­‐  

 

 

 

synostosis  have  been  known  for  some   time  [4,5],  only  recently  have  specific   mutations  in  a  number  of  genes   (FGFR3  and  MSX2  for  e.g.)  been  iden-­‐ tified  in  individuals  affected  with  non-­‐ syndromic  coronal  suture  synostosis   [5-­‐8].  The  number  of  familial  cases,   however,  remains  relatively  small  at   around  14%  for  coronal  cases  and  6%   for  sagittal  cases  [1,  9,10],  suggesting   that  sporadic  mutations  typically  are   involved.     While  significant  progress  has   been  made  in  elucidating  the  molecu-­‐ lar  causes  of  premature  suture  fusion,   our  understanding  of  the  disease  at   the  phenotypic  level  still  remains  in-­‐ complete.  In  particular,  it  is  unclear   how  alterations  at  the  genotypic  level   are  translated  into  the  range  of  varia-­‐

ble  phenotypes  that  present  under   the  heading  nonsyndromic  cranio-­‐ synostosis.  The  presence  of  certain   environmental  exposures  (e.g.  ciga-­‐ rette  smoke),  systemic  factors  (e.g.   circulating  levels  of  thyroxin  or  an-­‐ drogens)  and/or  the  additive  effects   of  modifier  genes  may  all  act  to  mod-­‐ ulate  phenotypic  severity  [1].  There-­‐ fore,  given  a  genetic  predisposition   for  nonsyndromic  craniosynostosis,   the  specific  phenotypic  outcome  may   depend  in  part  on  the  presence  of   additional  deleterious  factors,  both   intrinsic  and  extrinsic  to  the  organ-­‐ ism.  The  presence  of  such  factors  dur-­‐ ing  ontogeny  may  create  a  develop-­‐ mental  environment  characterized  by   amplified  stress,  leading  to  both  in-­‐ creased  synostotic  severity  and,  pos-­‐ sibly,  other  phenotypic  manifesta-­‐

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This  journal  is  published  by  the  University  Library  System,  University  of  Pittsburgh  as  part  of  its  D-­‐Scribe  Digital  Publishing  Program  and  is  cosponored   by  the  University  of  Pittsburgh  Press.  

Fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis    

Vol  5,  No  1  (2017)        DOI  10.5195/d3000.2017.78  

tions.  Unfortunately,  little  is  known   about  the  phenotypic  correlates  of   nonsyndromic  craniosynostosis  apart   from  the  well-­‐documented  secondary   growth  effects  on  the  skull.  However,   because  many  of  the  growth  factors   involved  in  the  regulation  of  suture   morphogenesis  have  expression  pat-­‐ terns  in  a  variety  of  other  tissues   throughout  the  developing  organism   [1,2-­‐5,11,12]  it  is  unlikely  that  the   extent  of  the  disruption  in  nonsyn-­‐ dromic  craniosynostosis  is  limited   entirely  to  the  suture  site.   Increased  stress  during  de-­‐ velopment,  either  of  genomic  or  envi-­‐ ronmental  origin,  has  been  linked  to   specific  phenotypic  patterns  in  organ-­‐ isms.  One  of  the  most  commonly   used  phenotypic  indicators  of  devel-­‐ opmental  stress  is  the  degree  of  ran-­‐ dom  asymmetry  present  in  normally   symmetrical,  paired  traits,  commonly   referred  to  as  fluctuating  asymmetry   [13].  Mather  [14]  reasoned  that  be-­‐ cause  bilateral  characteristics  are   programmed  by  the  same  genes,  ran-­‐ dom  deviations  from  symmetry  will   represent  a  phenotypic  record  of  the  

level  of  instability  present  during  de-­‐ velopment.  Waddington  [15,16]  used   the  term  canalization  to  refer  to  the   degree  to  which  genetically  pro-­‐ grammed  developmental  processes   are  able  to  resist  perturbations  during   ontogeny.  Developmental  instability   can  be  understood  as  the  degree  to   which  developmental  processes  are   unable  to  buffer  against  such  pertur-­‐ bations,  the  consequences  of  which   are  manifested  in  the  phenotype   [17,18].   Interest  in  developmental  in-­‐ stability  as  a  mechanism  for  elucidat-­‐ ing  the  complex  relationship  between   genotype  and  phenotype  has  in-­‐ creased  dramatically  over  the  last   decade  [18-­‐20].  Importantly,  elevated   levels  of  fluctuating  asymmetry  have   been  associated  with  a  variety  of   stressors  during  development,  includ-­‐ ing  a  number  of  genetic  diseases  [21].   For  example,  there  is  evidence  of  ele-­‐ vated  fluctuating  asymmetry  in  hu-­‐ mans  affected  with  orofacial  clefts   [22-­‐25],  Down’s  syndrome  [26,27],   Fragile-­‐X  syndrome  [28],  premature   birth  [29,30]  attention  deficit-­‐

Figure  1.  Superior  view  of  skulls  from  a  42  day  wild-­‐type  normal  control  rabbits  and  rabbits   with  late-­‐onset  (postnatal)  and  early-­‐onset  (prenatal)  coronal  suture  synostosis.  Note  the   shorter,  wider  cranial  vaults  in  the  rabbits  with  coronal  suture  synostosis  compared  to  the   wild-­‐type  rabbit  skull.  

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hyperactivity  disorder  [31],  general-­‐ ized  mental  retardation  [32],  spinal   malformation  [33]  and  a  wide  range   of  psychiatric  diseases  including   schizophrenia,  major  depression  and   bipolar  disorder  [21,34].  Because   nonsyndromic  craniosynostosis  is  a   disease  that  disrupts  craniofacial  de-­‐ velopment  through  abnormal  calvari-­‐ al  bone  homeostasis  resulting  in  ex-­‐ cessive  ossification  and  premature   suture  fusion  [2,3,35-­‐37],  affected   organisms  likely  experience  increased   systemic  stress  during  ontogeny.  In   principle,  such  stress  should  manifest   itself  in  the  phenotype  as  elevated   levels  of  fluctuating  asymmetry.   However,  De  Leon  and  Richtsmieir   [38]  reported  little  fluctuating  asym-­‐ metry  in  the  cranial  vault  bones  of   children  with  nonsyndromic  sagittal   suture  synostosis  and  deserves  fur-­‐ ther  study.  Given  the  overlap  in  mo-­‐ lecular  expression  between  calvarial   suture  and  dental  morphogenesis   [39],  the  association  between  dental   abnormalities  and  syndromic  forms  of   craniosynostosis  [40],  and  the  fact   that  both  syndromic  and  nonsyn-­‐ dromic  cases  may  share  a  common   genetic  basis  [5-­‐8],  it  is  reasonable  to   suspect  that  the  teeth  of  nonsyn-­‐ dromic  craniosynostotic  organisms   develop  within  a  stressed  physiologi-­‐ cal  environment  and  may  be  a  prime   candidate  for  manifesting  elevated   levels  of  fluctuating  asymmetry.  In   order  to  test  the  hypotheses  that  ge-­‐ nomic  stress  is  elevated  in  the  pres-­‐ ence  of  nonsyndromic  craniosynosto-­‐ sis,  and  that  such  stress  increases   with  the  severity  of  the  cranio-­‐ synostotic  phenotype,  we  propose  to   investigate  patterns  of  fluctuating   dental  asymmetry  in  a  rabbit  model   with  variably  expressed,  familial,  non-­‐ syndromic,  coronal  suture  synostosis   under  controlled  laboratory  condi-­‐ tions.  

Fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis    

Vol  5,  No  1  (2017)        DOI  10.5195/d3000.2017.78  

Materials  and  Methods   Study  sample   Our  study  sample  consisted  of  crani-­‐ odental  skeletal  material  from  176   New  Zealand  White  rabbits   (Oryctolagus  cuniculus)  affected  with   varying  degrees  of  familial,  nonsyn-­‐ dromic,  primary  coronal  suture   synostosis.  The  rabbits  were  part  of  a   unique  breeding  colony,  established   in  1993,  at  the  University  of  Pitts-­‐ burgh  [41-­‐46].  It  has  served  as  a  use-­‐ ful  model  for  the  study  of  familial,   nonsyndromic  craniosynostosis  in   human  infants  [35,  41-­‐47].  All  crania   were  obtained  from  rabbits  enrolled   in  ongoing  research  protocols.  All   protocols  were  reviewed  and  ap-­‐ proved  by  the  University  of  Pitts-­‐ burgh,  Institutional  Animal  Care  and   Use  Committee  (IACUC).   Rabbits  from  this  colony  ex-­‐ hibit  a  wide  range  of  phenotypes,  in-­‐ cluding:  phenotypically  normal  carri-­‐ ers;  rabbits  with  delayed-­‐onset   synostosis  (presenting  at  about  42   days  post-­‐natal),  and;  rabbits  with   early-­‐onset  complete  unilateral  or   bilateral  synostosis  (presenting  at   about  21  days  of  gestation)  [37,  41-­‐ 45]  (Figures  1  and  2).  It  has  been   shown  recently  that  rabbits  with  ear-­‐ ly-­‐onset  coronal  suture  synostosis   (i.e.,  the  most  severe  phenotype)   were  the  most  inbred  and  exhibited   the  highest  coefficients  of  inbreeding   [46].  For  the  present  study,  dentition   from  the  skulls  of  40  rabbits  with  ear-­‐ ly-­‐onset  synostosis,  65  rabbits  with   delayed-­‐onset  synostosis,  and  46  in-­‐ colony,  phenotypically  normal  rabbits   were  analyzed.  Further,  25  wild-­‐type   rabbits  of  the  same  breed  were  in-­‐ cluded  as  a  separate  control  group.   Table  1  shows  basic  descriptive  statis-­‐ tics  of  the  sample  by  group.  No  dif-­‐ ference  in  the  distribution  of  males   and  females  across  groups  was  ob-­‐  http://dentistry3000.pitt.edu  

served.    Thus,  rabbits  of  both   sexes  were  pooled  during   analysis.    Further,  due  to  sam-­‐ ple  size  limitations,  bilateral   and  unilateral  affected  animals   were  combined  for  all  anal-­‐ yses.  All  rabbits  were  housed   and  fed  under  similar  labora-­‐ tory  conditions.     Measurement  procedure     The  mesiodistal  (MD)   and  buccolingual  (BL)  dimen-­‐ sions  of  the  right  and  left  first   maxillary  molars  were  meas-­‐ ured  with  a  set  of  Mitotoyo   digital  calipers  (Japan)  (Figure   3).  The  first  maxillary  molar   was  chosen  because  evidence   suggests  it  is  a  very  stable   tooth  developmentally  [48].   This  conservative  approach   Figure  2.  Superior  view  of  the  coronal  suture  from  a   should  bias  our  results  against   42  day  wild-­‐type  normal  control  rabbits  and  rabbits   finding  fluctuating  asymmetry,   with  late-­‐onset  (postnatal)  and  early-­‐onset  (prena-­‐ thus  minimizing  the  chances  of   tal)  coronal  suture  synostosis.  Note  the  obliterated   reporting  a  false-­‐positive.  For  the   coronal  suture  in  the  early-­‐onset  synostosis  rabbit   skull  (bottom  picture),  the  dysmorphic  and  incom-­‐ MD  measurement,  the  arms  of   plete  coronal  suture  in  the  late-­‐onset  synostosis   the  calipers  were  positioned  at   rabbit  skull  (middle  picture),  compared  to  a  normal   the  widest  portion  of  the  tooth   coronal  suture  in  the  wild-­‐type  rabbit  skull  (top   at  the  occlusal  surface  (Figure  3).   picture).   For  the  BL  measurement,  one   arm  of  the  calipers  was  positioned     Following  standardized   flush  against  the  buccal  edge  of  the   methodological  guidelines  [49,50],   tooth.  With  the  one  arm  in  place,  the   the  statistical  nature  of  the  asym-­‐ measurement  was  then  taken  across   metry  was  explored  prior  to  further   the  tooth  at  the  level  just  below  the   analysis.  For  fluctuating  asymmetry,   occlusal  plane  on  the  lingual  edge   two  conditions  must  be  met.  The  raw,   (Figure  3).  The  BL  measurement  was   signed  left-­‐right  difference  scores   not  taken  directly  at  the  occlusal  sur-­‐ must  have  both:  1)  a  normal  distribu-­‐ face  in  order  to  avoid  measuring  the   tion  and  2)  a  mean  equal  to  zero.  The   effects  of  functional  wear.  All  meas-­‐ first  assumption  was  tested  with  the   urements  were  taken  by  one  individ-­‐ Kolmogorov-­‐Smirnov  test  for  normali-­‐ ual  (MAH).   ty,  while  a  one  sample  t-­‐test  was  per-­‐ formed  to  determine  whether  the   Statistical  analysis   mean  of  the  signed  asymmetry  scores     Testing  assumptions  of  fluc-­‐ is  significantly  different  from  zero.  A   tuating  asymmetry:   mean  significantly  different  from  zero   indicates  directional  asymmetry,   which  may  have  different  biological  

Fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis    

Vol  5,  No  1  (2017)        DOI  10.5195/d3000.2017.78  

implications  than  fluctuating  asym-­‐ metry.  Tests  of  fluctuating  asymmetry   assumptions  were  carried  out  for   each  variable  within  each  group  sepa-­‐ rately.     For  both  MD  and  BL  varia-­‐ bles,  the  phenotypically  normal  rab-­‐ bits  were  the  only  group  that  failed   the  test  of  normality  (p  <  0.05).  The   phenotypically  normal  (in-­‐colony)   control  group  was  therefore  dropped   from  subsequent  analyses.  For  the  BL   variable,  mean  raw  asymmetry  in  the   delayed-­‐onset  group  was  significantly   different  from  zero  (t  =  3.869;  p  <   0.001),  indicating  the  presence  of  di-­‐ rectional  asymmetry.  As  a  result,  the   delayed-­‐onset  group  was  dropped   from  further  analysis  of  the  BL  varia-­‐ ble.   Because  random  measure-­‐ ment  error  is  statistically  identical  to   fluctuating  asymmetry,  the  degree  of   error  present  in  the  data  must  also  be   assessed.  Repeat  measurements   were  obtained  on  all  176  animals  by   the  same  observer  (MAH).  For   each  variable,  measurement   error  was  quantified  by  compu-­‐ ting  the  mean  absolute  differ-­‐ ence  (MAD)  from  the  replicate   measures  [51]:   Mean  Absolute  Difference  =  ∑   |Time  1  –  Time  2|  /  N   This  value  was  then   compared  directly  against  the   standard  unsigned  mean  fluc-­‐ tuating  asymmetry  index:   Fluctuating  asymmetry  =  ∑   |Left  –  Right|  /  N   If  the  magnitude  of  the   mean  absolute  difference  is   determined  to  be  less  than  that   of  mean  fluctuating  asymmetry   for  a  given  variable,  this  indi-­‐ cates  that  residual  fluctuating    http://dentistry3000.pitt.edu  

asymmetry  exists  even  after  meas-­‐ urement  error  is  taken  into  account.   Conversely,  if  the  mean  absolute  dif-­‐ ference  is  greater  than  mean  fluctuat-­‐ ing  asymmetry,  then  we  cannot  be   certain  that  any  measurable  fluctuat-­‐ ing  asymmetry  exists.  For  both  varia-­‐ bles  in  the  present  study,  mean  abso-­‐ lute  difference  was  less  than  mean   fluctuating  asymmetry  (MD:  0.05  vs.   0.07;  BL:  0.04  vs.  0.09).  This  pattern   held  true  both  overall  and  within   each  group  separately,  indicating  that   fluctuating  asymmetry  was  not   swamped  by  random  measurement   error.   For  some  biological  traits,  in-­‐ creased  trait  size  is  related  to  elevat-­‐ ed  levels  of  fluctuating  asymmetry.   Thus,  the  effects  of  size  on  fluctuating   asymmetry  magnitude  must  be  con-­‐ sidered.  For  each  variable,  Pearson   correlation  coefficients  were  calcu-­‐ lated  between  original  trait  values   and  unsigned  FA  values  (|Left  –   Right|).  Because  a  weak  but  signifi-­‐

cant  correlation  (r  =  0.20;  p  <  0.01)   was  observed  for  the  MD  variable,  an   alternative  size-­‐adjusted  FA  index  was   used  for  subsequent  group  compari-­‐ sons:   Fluctuating  asymmetry  =  (|Left  –   Right|  /  0.5  Left  +  Right)     Group  fluctuating  asymmetry   comparisons:   Using  the  standard  size-­‐ adjusted  index,  mean  fluctuating   asymmetry  across  groups  was  com-­‐ pared  using  nonparametric  statistics,   followed  by  post-­‐hoc  comparisons   where  necessary.  The  nonparametric   approach  was  employed  because  the   fluctuating  asymmetry  index  chosen   for  group  comparisons  converts  left-­‐ right  differences  into  absolute  values,   which  by  nature  are  not  distributed   normally.  Because  certain  groups   failed  to  meet  the  assumptions  of   fluctuating  asymmetry,  certain  group   comparisons  could  not  be  carried  out.   For  the  MD  variable,  only  three  

Fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis    

Vol  5,  No  1  (2017)        DOI  10.5195/d3000.2017.78  

groups  (early-­‐onset,  delayed-­‐onset,   and  wild-­‐type  controls)  were  com-­‐ pared  with  a  Kruskal-­‐Wallis  ANOVA.   For  the  BL  variable,  only  two  groups   (early-­‐onset  and  wild-­‐type  controls)   were  compared,  thus  a  Mann-­‐ Whitney  U  t-­‐test  was  used.  Statistical   analyses  were  performed  with  the   program  SPSS  11.5.  All  differences   were  considered  significant  if  p  <   0.05.  

Results     For  the  MD  dimension,  rab-­‐ bits  with  early-­‐onset  synostosis  had   more  fluctuating  asymmetry  than   rabbits  with  delayed-­‐onset  synostosis   while  wild-­‐type  control  rabbits  had  a   greater  magnitude  of  fluctuating   asymmetry  than  either  affected   group.  However,  no  significant  (p  >   0.05)  group  differences  in  mean  fluc-­‐ tuating  asymmetry  were  observed  for   the  MD  dimension  (Table  2).     In   contrast  for  the  BL  dimension,  rabbits   with  complete  early-­‐onset  synostosis   had  a  significantly  (p  <  0.05)  greater   magnitude  of  fluctuating  asymmetry   than  wild-­‐type  control  rabbits  (Table   2).  Although  mean  age  differed  con-­‐

 http://dentistry3000.pitt.edu  

siderably  across  groups,  no  significant   correlation  (p>  0.05)  between  FA  and   age  was  observed  for  either  tooth   dimension.      

Discussion     Familial,  nonsyndromic  crani-­‐ osynostosis  is  a  disease  characterized   by  abnormal  calvarial  bone  regulation   early  in  prenatal  life  [2,3,35-­‐37].  It   was  hypothesized  that  this  disruption   in  homeostasis  would  lead  to  in-­‐ creased  systemic  physiological  stress   during  the  critical  period  of  den-­‐ togenesis,  resulting  in  increased  fluc-­‐ tuating  dental  asymmetry.  Results  of   the  present  study  confirm  this  hy-­‐ pothesis;  rabbits  with  early-­‐onset   synostosis  had  a  significantly  greater   magnitude  of  fluctuating  dental   asymmetry  for  the  BL  dimension  of   the  first  maxillary  molars  compared   to  unaffected  controls.  This  pattern   was  not  observed,  however,  for  the   MD  dimension.  These  results  suggest   that  the  teeth  of  rabbits  with  congen-­‐ ital  nonsyndromic  craniosynostosis   develop  within  a  stressed  physiologi-­‐

cal  environment.  Unfortunately,  be-­‐ cause  certain  groups  did  not  satisfy   the  statistical  requirements  of  fluctu-­‐ ating  asymmetry,  all  group  compari-­‐ sons  were  not  possible.  It  could  not   be  determined,  therefore,  whether   fluctuating  asymmetry  was  related  to   craniosynostosis  in  a  severity-­‐ dependent  manner.  These  results,   although  limited  in  scope,  suggest   that  a  broader  array  of  primary  de-­‐ fects  may  be  present  in  individuals   with  nonsyndromic  craniosynostosis,   and  that  these  may  not  be  limited   entirely  to  the  suture  site.  In  princi-­‐ ple,  a  fuller  understanding  of  the   phenotypic  correlates  present  in  non-­‐ syndromic  craniosynostosis  may  shed   light  on  disease  etiology.  For  in-­‐ stance,  predictions  can  be  generated   about  patterns  of  phenotypic  varia-­‐ tion  from  the  known  expression  pat-­‐ terns  of  specific  candidate  genes.     Prior  to  the  present  study,   dental  fluctuating  asymmetry  had  not   been  examined  in  individuals  affected   with  craniosynostosis.  Previous  re-­‐ search  into  dental  development  with-­‐ in  the  craniosynostoses  has  been  fo-­‐ cused  almost  entirely  on  syndromic   forms  of  the  disease.  In  Apert  syn-­‐ drome,  for  instance,  a  wide  variety  of   dental  anomalies  have  been  report-­‐ ed;  these  include  delayed  develop-­‐ ment,  ectopic  teeth,  hypodontia  and   supernumerary  teeth  [52,53].  Similar-­‐ ly,  in  cases  of  Crouzon  syndrome,  ec-­‐ topic  eruption,  delayed  development   and  hypodontia  have  all  been  ob-­‐ served  [54,55,12].  Pfeiffer  syndrome   may  be  characterized  by  natal  teeth   [56],  suggesting  precocious  dental   development.  This  is  further  support-­‐ ed  by  reports  of  supernumerary  and   ectopic  teeth  [57].  Given  the  fact  that   FGFs  play  a  role  in  both  dental  devel-­‐ opment  and  suture  morphogenesis,  it   is  perhaps  not  surprising  that  the   teeth  are  affected  in  cases  of  syn-­‐

Fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis    

Vol  5,  No  1  (2017)        DOI  10.5195/d3000.2017.78  

dromic  craniosynostosis  [39,11,58].  In   light  of  this  overlap  in  molecular  ex-­‐ pression,  these  conspicuous  dental   defects  are  most  likely  the  result  of   aberrant  FGF  signaling  within  the  de-­‐ veloping  dentition  and  its  surround-­‐ ing  tissues  [40].    

in  a  number  of  naturally  and  experi-­‐ mentally  stressed  human  and  non-­‐ human  populations,  although  the   source  of  the  stress  is  sometimes  not   well  defined  [24,  27,  28,  64-­‐70].    

A  number  of  possible  expla-­‐ nations  for  the  present  findings  may   It  remains  unclear,  however,   be  offered.    One  possibility  is  that  the   whether  the  observed  pattern  of  den-­‐ same  genetic  mutation  that  causes   tal  fluctuating  asymmetry  results   premature  suture  fusion  also  results   from  such  pleiotropic  effects.  Be-­‐ in  increased  dental  fluctuating  asym-­‐ cause  fluctuating  asymmetry  is  a  ra-­‐ metry.  The  genetic  mutation  in  these   ther  generic  response  to  stress  during   rabbits  has  been  identified  recently  as   development,  we  can  only  speculate   belonging  to  the  fgfr  family  of  genes   as  to  whether  the  underlying  cause  of   working  in  tandem  with  a  modifying   gene  (manuscript    in   preparation)  so  this   may  be  a  possibility;   however,  no  obvious   dental  anomalies   have  been  observed   in  this  model,  sug-­‐ gesting  that  overall   dentogenesis  is   probably  not  affect-­‐ ed  in  these  rabbits.     Accumulating  evi-­‐ dence  also  suggests   that  altered  calvarial   bone  regulation,   Figure  3.    Inferior  view  of  the  rabbit  skull  showing  the  post-­‐ resulting  in  hyperos-­‐ incisor  maxillary  dentition  (P1-­‐M3)  (first  three  premolars  and   tosis  and  subsequent   molars).    The  arrows  on  M1  (first  molar)  represent  the  mesi-­‐ premature  suture   odistal  and  buccolingual  dimensions  that  were  measured  in  the   fusion  in  cases  of   present  study.   nonsyndromic  crani-­‐ the  fluctuating  asymmetry  is  the   osynostosis,  stems  from  localized,   same  as  that  of  the  craniosynostosis.   abnormal  expression  of  various  bone   Dental  dimensions  have  been  shown   regulating  growth  factors  (e.g.,  BMPs,   to  be  highly  heritable  and  are  thought   FGFs,  and/or  TGFβs)  [1-­‐3,  36].  Such   to  be  under  relatively  tight  genetic   abnormal  growth  factor  expression   control  [59-­‐61].  That  being  said,  it  is   may  also  primarily  affect  other  devel-­‐ also  clear  that  environmental  influ-­‐ oping  antimeric  osseous  structures   ences  such  as  local  and  systemic   resulting  in  fluctuating  asymmetry.   physiological  factors,  teratogens,  and   However,  DeLeon  and  Richtsmieir   extra-­‐organismic  variables  play  a  crit-­‐ [38]  reported  no  significant  fluctuat-­‐ ical  role  in  determining  the  final  form   ing  asymmetry  in  the  crania  of  22   and  size  of  teeth,  especially  during   children  with  nonsyndromic  sagittal   the  early  stages  of  dental  develop-­‐ synostosis,  although  the  effects  could   ment  [62,63].  Increased  fluctuating   have  been  ameliorated  with  age  and   dental  asymmetry  has  been  reported    http://dentistry3000.pitt.edu  

remodeling.  In  addition,  no  significant   changes  in  postcranial  skeletal  (e.g.,   first  metacarpal)  growth  have  been   observed  in  synostotic  rabbits  [41-­‐ 45],  although  fluctuating  skeletal   asymmetry  has  not  been  examined   yet  and  this  mechanism  remains  a   possibility.     Another  possibility  is  that  in-­‐ creased  intracranial  pressure  result-­‐ ing  from  premature  suture  fusion   could  invoke  a  generalized  stress  re-­‐ sponse  by  activating  the  hypothalam-­‐ ic-­‐pituitary-­‐adrenal  axis,  which  may   increase  levels  of  fluctuating  calcium   during  dentogenesis  [65,71],  and  sec-­‐ ondarily  result  in  increased  dental   fluctuating  asymmetry  in  rabbits  with   early-­‐onset  synostosis.  Increased  fluc-­‐ tuating  asymmetry  has  also  been   linked  to  external  environmental   stressors  such  as  heat,  cold  and  noise   [64,65,67].  However,  these  factors   are  unlikely  to  play  a  major  role  in   this  study  because  all  the  rabbits  in   the  present  sample  were  raised  under   similar  controlled,  environmental   conditions.  It  is  also  unlikely  that   functional  wear  effects  account  for   these  results  since  such  effects  are   typically  associated  with  directional   asymmetries  and  should  increase   with  age  (no  association  between  age   and  fluctuating  asymmetry  was  ob-­‐ served).  In  addition,  rabbit  teeth  are   continually  erupting  and  the  lack  of   age  related  differences  in  dental  fluc-­‐ tuating  asymmetry  suggest  that  the   stress  associated  with  craniosynosto-­‐ sis  may  be  chronic  in  rabbits  with  ear-­‐ ly-­‐onset  synostosis.    Finally,  canaliza-­‐ tion  of  dental  development  may  be   under  separate  genetic  control.  Un-­‐ der  this  premise,  less  canalized  organ-­‐ isms  would  be  predisposed  to  mani-­‐ festing  elevated  dental  fluctuating   asymmetry  in  the  presence  of  delete-­‐ rious  factors,  such  as  aberrant  growth   factor  signaling.  We  recently  reported  

Fluctua'ng  dental  asymmetry  in  rabbits  with  familial  nonsyndromic  coronal  suture  synostosis    

Vol  5,  No  1  (2017)        DOI  10.5195/d3000.2017.78  

a  significant,  negative  relationship   between  coronal  suture  growth  and   the  coefficient  of  inbreeding  [46]  in   rabbits  from  this  colony.  Rabbits  with   early  onset  synostosis  showed  the   least  growth  and  greatest  amount  of   inbreeding.  It  is  possible  that  addi-­‐ tional  modifier  genes  accumulated   from  inbreeding  were  responsible  for   increased  systemic  stress  and  the  in-­‐ creased  levels  of  fluctuating  dental   asymmetry  in  these  rabbits.  Hueze  et   al.  [72]  also  showed  that  human  cra-­‐ nia  exhibiting  a  phenotypic  continu-­‐ um  with  skulls  with  bicoronal  sutures   synostosis  displaying  more  intense   asymmetry  that  skulls  with  unicoro-­‐ nal  shape  which  supports  this  hy-­‐ pothesis.   Unfortunately,  the  present   study  was  not  designed  to  test  these   competing  hypotheses.  Fluctuating   asymmetry  is  a  subtle  marker  of  de-­‐ velopmental  disturbance  and  its  per-­‐ vasiveness  both  in  the  craniodental   complex  and  other  anatomical  re-­‐ gions  of  craniosynostotic  organisms   will  need  to  be  explored  further  in   order  to  begin  to  unravel  its  biological   basis.    Given  the  numerous  reports  of   various  dental  defects  associated   with  syndromic  craniosynostosis,  it   may  be  worthwhile  to  investigate   other  aspects  of  the  dentition  in  non-­‐ syndromic  synostotic  populations  as   well.   Acknowledgements   The  authors  would  like  to   thank  Kate  Quinlin  for  her  assistance   in  the  lab.  This  work  was  supported  in   part  by  a  grant  from  NIH/NIDCR  (P60   DE13078).  

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